Institute of Biomedical Sciences, Academia Sinica, Taipei, 11529, Taiwan.
Institute of Pharmacology, National Yang-Ming University, Taipei, 11221, Taiwan.
Breast Cancer Res. 2017 Oct 5;19(1):111. doi: 10.1186/s13058-017-0904-8.
Core fucosylation (addition of fucose in α-1,6-linkage to core N-acetylglucosamine of N-glycans) catalyzed by fucosyltransferase 8 (FUT8) is critical for signaling receptors involved in many physiological and pathological processes such as cell growth, adhesion, and tumor metastasis. Transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT) regulates the invasion and metastasis of breast tumors. However, whether receptor core fucosylation affects TGF-β signaling during breast cancer progression remains largely unknown.
In this study, gene expression profiling and western blot were used to validate the EMT-associated expression of FUT8. Lentivirus-mediated gain-of-function study, short hairpin RNA (shRNA) or CRISPR/Cas9-mediated loss-of-function studies and pharmacological inhibition of FUT8 were used to elucidate the molecular function of FUT8 during TGF-β-induced EMT in breast carcinoma cells. In addition, lectin blot, luciferase assay, and in vitro ligand binding assay were employed to demonstrate the involvement of FUT8 in the TGF-β1 signaling pathway. The role of FUT8 in breast cancer migration, invasion, and metastasis was confirmed using an in vitro transwell assay and mammary fat pad xenograft in vivo tumor model.
Gene expression profiling analysis revealed that FUT8 is upregulated in TGF-β-induced EMT; the process was associated with the migratory and invasive abilities of several breast carcinoma cell lines. Gain-of-function and loss-of-function studies demonstrated that FUT8 overexpression stimulated the EMT process, whereas FUT8 knockdown suppressed the invasiveness of highly aggressive breast carcinoma cells. Furthermore, TGF-β receptor complexes might be core fucosylated by FUT8 to facilitate TGF-β binding and enhance downstream signaling. Importantly, FUT8 inhibition suppressed the invasive ability of highly metastatic breast cancer cells and impaired their lung metastasis.
Our results reveal a positive feedback mechanism of FUT8-mediated receptor core fucosylation that promotes TGF-β signaling and EMT, thus stimulating breast cancer cell invasion and metastasis.
核心岩藻糖基化（在 N-糖链的核心 N-乙酰葡萄糖胺的α-1,6-连接上添加岩藻糖）由岩藻糖基转移酶 8（FUT8）催化，对于涉及细胞生长、黏附和肿瘤转移等许多生理和病理过程的信号受体至关重要。转化生长因子-β（TGF-β）诱导的上皮-间充质转化（EMT）调节乳腺癌的侵袭和转移。然而，受体核心岩藻糖基化是否影响乳腺癌进展过程中的 TGF-β 信号仍知之甚少。
在这项研究中，使用基因表达谱分析和 Western blot 来验证 FUT8 与 EMT 相关的表达。使用慢病毒介导的功能获得研究、短发夹 RNA（shRNA）或 CRISPR/Cas9 介导的功能丧失研究以及 FUT8 的药理学抑制来阐明 FUT8 在乳腺癌细胞中 TGF-β 诱导的 EMT 中的分子功能。此外，使用凝集素印迹、荧光素酶测定和体外配体结合测定来证明 FUT8 参与 TGF-β1 信号通路。使用体外 Transwell 测定和体内乳腺脂肪垫异种移植肿瘤模型来证实 FUT8 在乳腺癌迁移、侵袭和转移中的作用。
基因表达谱分析显示，FUT8 在 TGF-β 诱导的 EMT 中上调；该过程与几种乳腺癌细胞系的迁移和侵袭能力有关。功能获得和功能丧失研究表明，FUT8 的过表达刺激 EMT 过程，而 FUT8 的敲低则抑制高侵袭性乳腺癌细胞的侵袭能力。此外，TGF-β 受体复合物可能被 FUT8 核心岩藻糖化以促进 TGF-β 结合并增强下游信号。重要的是，FUT8 抑制抑制了高度转移性乳腺癌细胞的侵袭能力并损害了它们的肺转移。
我们的结果揭示了 FUT8 介导的受体核心岩藻糖基化促进 TGF-β 信号和 EMT 的正反馈机制，从而刺激乳腺癌细胞的侵袭和转移。
